Organisms have evolved mechanisms to control cellular processes by the addition and removal of phosphate groups to and from molecules. Organisms use phosphorylation and dephosphorylation to transmit and integrate signals from their environment. For example, a pollen grain landing on a stigma leads to a series of protein phosphorylation events that ultimately triggers the onset of fertilization. In addition, phosphorylation is a mechanism for cellular regulation of processes such as cell division, cell growth and cell differentiation. In proteins, a phosphate group can modify serine, threonine, tyrosine, histidine, arginine, lysine, cysteine, glutamic acid and aspartic acid residues.
The emerging field of proteomics deals with the characterization and regulation of proteins in organisms. Since phosphorylation plays such a major role in protein regulation, there is a need for an accurate, fast, simple and inexpensive method to measure the degree of phosphorylation in samples.
Currently, the methods used to detect phosphorylation in a sample include fluorescent assays, radioimmunoassays, immobilized metal affinity chromatography (IMAC), two-dimensional electrophoretic gel separation (2-D PAGE) coupled with mass spectrometry detection, and liquid chromatography separation coupled with mass spectrometry detection. Normally detection of the mass spectrometer requires electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI). Methods for quantitation of phosphate include 31P nuclear magnetic resonance spectroscopy (NMR) and radioisotope (γ-32P) labelling with scintillation counting or multiphoton (MPD) detection (Godovac-Zimmerman and Brown, 2001).
However, the above methods are either laborious or hindered by the need to use radioisotopes, and are not considered quantitative in that they do not precisely determine the number of phosphate groups in the sample. These methods require a two-step process, whereby a portion of the sample is used to determine the phosphorous concentration, and another portion of the sample is used to determine the concentration of the sample. Thus, there is variability in the degree of sample utilization. For example, if one is assaying a protein sample, quantitation of the protein is required to accurately assess the number of phosphorylation sites per protein molecule. This measurement requires a completely different assay done independently of phosphate detection. The only truly accurate method for determining protein concentration is to acid hydrolyse a portion of the sample and then perform amino acid analysis on the hydrolysate (Wilson and Walker, 2000). Other approximate methods rely on the presence of particular amino acid residues in the proteins. For example, tyrosine and tryptophan are measured in ultraviolet absorption, and arginine and lysine are measured in the Bradford™ calorimetric assay. The Kjeldahl analysis measures total nitrogen, and the far ultraviolet absorption method is based on the number of peptide bonds. However, these assays are relative as the concentration of particular amino acid residues and even nitrogen in proteins varies significantly (Wilson and Walker, 2000).
Recently, Wind et al. (2001) disclosed a method for determining the degree of protein phosphorylation by the simultaneous detection of phosphorus and sulfur ions using inductively coupled plasma mass spectrometry (ICP-MS). However, this method required ‘adjustments’ to avoid or minimize isobaric interference. For example, a very slow flow rate was required (4 microlitre/minute) to minimize isobaric interferences from solvent molecules. The method required the use of ‘high resolution’ ICP-MS, which is a very expensive instrument and generally is not used for routine measurements. These restrictions make the method laborious and expensive.
Thus, the current methods to determine the degree of phosphorylation of a sample either (i) lack accurate quantitation, (ii) require a two-step process, or (iii) they require a very expensive machine.
The use of ICP-MS to detect phosphorus and quantify the degree of phosphorylation in samples is hindered by the low degree of ionization of phosphorus (33%) and the elements that can characterize a biological sample namely O, N, H (0.1%), C (5%), S (14%). The degrees of ionization given here in the brackets are taken from Houk, R. S., 1986. Another limitation is the presence of high background of these ions that originate from the plasma ion source, sample matrix or the vacuum system of the instrument, and also the presence of spectral interferences of other atomic or polyatomic ions at the same mass of the ions of interest. For example, 31P+ is interfered by the presence of 15N 16O+, 14N17O+, 14N16OH+ and others. The latter limitation is referred to as isobaric interference.
U.S. Pat. No. 6,140,638 to Tanner and Baranov, discloses a reactive collision cell used in conjunction with an inductively coupled plasma mass spectrometer (ICP-MS), to reduce isobaric interference, by reactive removal of the interference, in which a dynamic bandpass is employed to reject intermediate ions which would otherwise react to form new isobaric interferences. Recently, this reactive collision cell has also been used to produce secondary ions or product ions with a different mass to charge ratio than the interfering ions, to minimize isobaric interference (Baranov and Tanner, 1998; Tanner and Baranov, 1998; Baranov and Tanner, 1999; Tanner and Baranov, 1999; Bollinger and Schleisman, 1999).
WO 01/01446 to Todd et al., 2000 discloses a similar approach to minimize isobaric interference using a different apparatus. Similarly, Eiden et al., 1997, discloses the use of an Ion Trap Mass Spectrometer coupled to an octopole ion guide/collision cell to avoid isobaric interference, by using reactions to remove interferences to a different mass. Both these references use different instruments and do not deal with the detection and measurement of the degree of phosphorylation in samples.
With the great potential offered by the emerging field of proteomics, there is a need for the detection and measurement of the degree of phosphorylation in biological samples. There is a need for rapid method for quantitation, employing simultaneous measurement of the concentration of phosphorus and the concentration of the sample. Further, there is a need for a rapid and accurate confirmation of the results. Further still, there is a need for a simple and cheap method, requiring a small and relatively inexpensive machine.